Isolation, Purification And Characterization Of Acide Phosphatase From Sperm Of PIC Pigs (PIC344)

Acid phosphatase(ACPase,E.C.3.1.3.2) was isolated and purified from the sperm of PIC pigs(PIC344) and its biochemical properties was studied. The purified Acid phosphatase was obtained by ammunium sulfate fractionation, DEAE-Sepharose F. F ion-exchange column,and gel filtration with Sephacryl S-200. The purified enzyme moved as a single electrophoretic band in PAGE and SDS-PAGE. The final purified multiple was 22. 78 and the specific activity was 15. 26Umg. Pr with pNPP as its substrate. The optimum pH value for the enzyme was 3. 6. The optimum temperature for the enzyme was 52. ACPase was stable within pH 4.5~6.0 and its activity was stable below 40. The activity remained 59.2% after the enzyme cultured for 30min at 50 The Michealis-Menten constant {Km) was 3. 8 X 10-3mol/L on the pNPP as its substrate.It' s subunit weight was 52. 3kDa determined with SDS-PAGE. Isoelectric focusing study showed that pI of the enzyme was 5.11.The enzyme could been activated by K+, Mg2+, Ca2+, Ni2+ Mn2+, while inhibited by the ions of Cu2+ Cd2+ Fe2+, Zn2+ which concentration ranged from 1mmol/L to 5mmol/L ACPase and EDTA was inactivited by urea. The ability of metal ions activate the ACPase one by one in order for Mg2+> Ni2+> Mn2+> K+> Ca2+. The ability of metal ions inactivate the ACPase one by one in order for Cu2+> Cd2+> Fe2+> Zn2+ Cu2+ Cd2+ was selected for determing types of inhibition and the result showed that Cu2+ , Cd2+ wasnoncompetitive inhibitors by using Lineweaver-Burk method and their Ki values were determined by Dixon method. Their Ki values espectively were 0. 58 X 10-3mmol/L 9. 44X 10-3mmol/L, The Fluorescent spectras of Mg2+ Ca2+ Cd2+ Cu2+ indicate that Mg2+ and Ca2+ caused the fluorescent spectrum strength to go up, Cd2+ and Cu2+ raise the fluorescent spectrum strength to decrease, the maximum exit wave-length was constant. The result explains that these metal ions influence the conformation of ACPase .The enzyme was modified by NBS, PMSF, pCMB, DTT and AcBr. The effects of different concentration and time on the ACPase catalytic activity were determined. It was found that the reactions of ACPase from PCI344 with NBS, PMSF, pCMB and DTT resulted in a strong inhibition of enzyme activities. The activities of ACPase decreased steadily with the increase of modification reagents concentration and time. Histidine residues modified by BrAc did not change the activity of the enzyme, while the activity only remained 18% after PMSF modified ACPase for 60min. These results predict that Trp, Ser and 2-SH group are essential for the activities of ACPase from PCI344. It also showed that His accouts for little to the activities of ACPase from PCI344.